Paxillin is a multi-domain, 68 KDa phosphoprotein that localizes to specialized actin-membrane-extracellular matrix attachment sites in cultured mammalian cells and in vivo. We will test the hypothesis that paxillin functions as a scaffold/adapter protein at the plasma membrane to coordinate the interplay between adhesion and growth factor-derived signals that result in reorganization of the actin cytoskeleton and control cell migration. Aim 1 will examine the importance of the interaction between paxillin and the PKL-PIX-PAK (p21-activated kinase)-Nck complex, focusing on its role in effecting PAK function. The critical role of adhesion and growth factor regulated PKL tyrosine phosphorylation will also be determined. Aim 2 will utilize paxillin null and PTP-PEST null fibroblasts to examine the essential role for paxillin-PTP-PEST interactions in controlling PTP-PEST-mediated effects on cell spreading, motility and p21-GTPase signaling. Aim 3 will determine how the differential expression of two paxillin related proteins, Hic-5 and paxillin delta modulate integrin signaling to paxillin and associated proteins to effect cell migration and morphology during epithelial-mesenchymal transition. To accomplish these Aims cytoskeletal organization will be evaluated by immunofluorescence microscopy. Time-lapse video microscopy, Boyden and Dunn chamber assays will permit evaluation of dynamic cell shape and motility changes. Phosphorylation specific antibodies will be used to identify changes in the activity and location of focal adhesion and cytoskeletal proteins as well MAP kinase and other signaling intermediates. Rho-family GTPases activities will be assessed using GST-GTPase effector protein binding domains. It is anticipated that information gained from the proposed study will provide insight into the molecular mechanisms by which cells interact and communicate with their environment to regulate normal and neoplastic cell migration and will potentially identify protein-protein interactions/pathways that might in the future serve as targets for therapeutic intervention.